Production and Characterization of a Camelid Single Domain Antibody–Urease Enzyme Conjugate for the Treatment of Cancer

Abstract

A novel immunoconjugate (L-DOS47) was developed and characterized as a therapeutic agent for tumors expressing CEACAM6. The single domain antibody AFAIKL2, which targets CEACAM6, was expressed in the Escherichia coli BL21 (DE3) pT7-7 system. High purity urease (HPU) was extracted and purified from Jack bean meal. AFAIKL2 was activated using N-succinimidyl [4-iodoacetyl] aminobenzoate (SIAB) as the cross-linker and then conjugated to urease. The activation and conjugation reactions were controlled by altering pH. Under these conditions, the material ratio achieved conjugation ratios of 8-11 antibodies per urease molecule, the residual free urease content was practically negligible (<2%), and high purity (>95%) L-DOS47 conjugate was produced using only ultradiafiltration to remove unreacted antibody and hydrolyzed cross-linker. L-DOS47 was characterized by a panel of analytical techniques including SEC, IEC, Western blot, ELISA, and LC-MS(E) peptide mapping. As the antibody-urease conjugate ratio increased, a higher binding signal was observed. The specificity and cytotoxicity of L-DOS47 was confirmed by screening in four cell lines (BxPC-3, A549, MCF7, and CEACAM6-transfected H23). BxPC-3, a CEACAM6-expressing cell line was found to be most susceptible to L-DOS47. L-DOS47 is being investigated as a potential therapeutic agent in human phase I clinical studies for nonsmall cell lung cancer.

Hypoxia and acidosis: immune suppressors and therapeutic targets

“…Ureases are urea‐degrading enzymes that convert urea into more toxic ammonia and lead to a local pH rise from the generated ammonia . CEACAM6‐targeted Jack bean urease (L‐DOS47) has begun to be used in preclinical tumour models and clinical trials to modify the tumour microenvironment …”

“…89 CEACAM6-targeted Jack bean urease (L-DOS47) has begun to be used in preclinical tumour models and clinical trials to modify the tumour microenvironment. 90 There are numerous possibilities to target tumour acidity more indirectly by influencing metabolism or ion transport. For example, carbonic anhydrase IX (CA-IX) controls intracellular and extracellular acid-base balance to maintain survival and is a key regulator of extracellular acidity.…”

Development and Characterization of a Camelid Single Domain Antibody–Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2

“…This was determined by interrogating the intensities of the five bands in the main cluster [see Ref. ( 9 ) for further details]. All CRs reported are average values.…”

“…Unlike most of the anti-angiogenic agents that interrupt the kinase signaling cascade by blocking the dimerization of VEGFR2 or by inhibiting kinase activity, our ADC, V21-DOS47, kills VEGFR2-expressing cells by inducing cytotoxic activity at the target cells. Similar to our previous antitumor immunoconjugate, L-DOS47 ( 9 ), V21-DOS47 is composed of a camelid single domain antibody and the enzyme urease (derived from jack beans, Canavalia ensiformis ): the V21 antibody binds to VEGFR2, thus targeting the complex to VEGFR2-expressing cells, whereas the urease enzyme converts endogenous urea into ammonia in situ to induce cytotoxicity. Since VEGFR2 is not only expressed in the tumor vasculature but has also been identified on the surface of a variety of tumors ( 4 , 10 , 11 ), V21-DOS47 targets both VEGFR2 + vascular endothelial cells and VEGFR2 + tumor cells.…”

“…This provides a challenge to large-scale production. In our previous report, we described conjugation chemistry and separation procedures designed to address these challenges ( 9 ). In this study, we evaluated additional antibody production and conjugation chemistry methods to generate a novel antibody–urease conjugate, V21-DOS47.…”